PCR principle, requirement and mechanism

Polymerase chain reaction (PCR) technique :

In 1983, American biochemist Kary Mullis described the technique of in vitro (in a test tube rather than an organism) gene amplification and named it as a Polymerase Chain Reaction (PCR). The PCR is a process of in vitro amplification (i.e. identical-multiplication) of genes of interest, and it is based on enzymatic replication of genetic material. The PCR is a temperature-dependent process of DNA amplification. The machine used in the PCR technique is known as a Thermo-cycler or Thermal-cycler or simply PCR machine. This machine provides the constant temperature and even rapidly changes between two temperature states within a very short time for each step to complete. In PCR technique the DNA molecule is cut horizontally (i.e. the H-bonds between the nucleotides of the opposite strands are broken). PCR can generate a billion copies of the desired segment of genetic material (i.e. DNA or RNA), with high accuracy and specificity, in a very short time period. One complete cycle of PCR takes around 3 to 4 minutes. The process of PCR is completely automated and involves automatic thermal cycles for denaturation and renaturation of double stranded DNA molecules. Thus, at the end of n cycles of PCR 2n copies of DNA segments are produced.

Amplification = 2n

Where; n - no. of PCR cycle.

Requirements for PCR :

The requirements for PCR includes;

• DNA template :

DNA containing the desired segment to be amplified.

• Oligonucleotides primers :

A primer is oligonucleotides (i.e. a short nucleic acid sequence) that provides a starting point for DNA synthesis. In PCR technology, a pair of primers is used to hybridise complementary sequences of DNA, viz; Forward primer, and Backward (or rivers) primer.

• Polymerase enzymes :

This enzyme is also called Replicase, when it replicates the DNA molecules. Polymerases are enzymes that catalyse (i.e. accelerate) the synthesis polynucleotide chain from 3' end to 5' end. DNA polymerases carry out the process of addition of nucleotides and formation of polynucleotide chains. Whose sequence is complementary to the original template. Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after the extremophilic bacteria (heat-tolerant bacterium) from which it was isolated called Thermus aquaticus.

• Deoxynucleotide triphosphates (dNTPs) :

Deoxynucleotide triphosphate (dNTPs) are the building blocks of genetic material (i.e. DNA or RNA). The members of dNTPs includes; dATP (deoxyadenosine triphosphate), dTTP (deoxythymidine triphosphate), dCTP (deoxycytidine triphosphate), and dGTP (deoxyguanosine triphosphate).

• Buffer solution : 

It provides a suitable chemical environment.

• Cofactors : 

Cofactors include; Divalent cations (Mg+2), and Monovalent ions (Potassium ions).

Mechanism of PCR :

At the start of PCR technique, all the requirements of PCR are mixed together in a tube called Eppendorf tube or PCR tube. The eppendorf tube with a mixture of PCR is kept in a PCR machine and then the process of polymerase chain reaction is started. The process of polymerase chain reaction is complete in three steps, viz;

• Step-I : Denaturation :

At this step the reaction mixture is heated to a temperature 90 to 98 oC. Due to heating of reaction mixture till here, the H-bonds between the nucleotides of opposite stands are broken. And therefore the desired DNA stand is separated.

• Step-II : Annealing :

At this step the reaction mixture is allowed to cool to a temperature 40 to 60 oC. Due to cooling of reaction mixture till here, the separated strands of desired DNA make H-bonds with primer and join together to the  complementary sequence on it. That provides a starting point for DNA synthesis.

• Step-III : Primer extension or Polymerization :

At this step the reaction mixture is again heated to a temperature 70 to 75 degree Celsius. Due to heating of reaction mixture again till here, the thermostable Taq DNA polymerase uses the single stranded DNA as a template and adds nucleotides.

After completion of all these steps in one cycle, again the second cycle is repeated following the same process. One complete cycle of PCR takes around 3 to 4 minutes. The process of PCR is completely automated and involves automatic thermal cycles for denaturation

and renaturation of double stranded DNA molecules. Thus, at the end of n cycles of PCR 2n copies of DNA segments are produced. After the desired number of cycles is completed, the amplified DNA segment is purified by gel electrophoresis. After sequencing of the desired gene, the amplified DNA segment can be inserted into a cloning vector.

Applications of PCR :

The following are the major applications of PCR in different fields, viz;

• IN MEDICINE :

Testing genetic disease mutations. Monitoring the gene in gene therapy. Detecting disease-causing genes in the parents.

• IN FORENSIC SCIENCE :

Used as a tool in genetic fingerprinting. Identifying the criminal from millions of people. Paternity tests. 

• IN RESEARCH AND GENETIC :

Compare the genome of two organisms in genomic studies. In the phylogenetic analysis of DNA from any source such as fossils. Analysis of gene expression. Gene Mapping.

Disadvantage of PCR machine :

One of the major disadvantages of a PCR machine or Thermo-cycler or Thermal-cycler is that, the PCR machine can raise temperature upto 100 oC but after that it is not able to lower the temperature below 70 oC automatically. If a PCR machine fails to lower the temperature below 70 oC then the annealing step of PCR would be affected. If annealing does not take place then ultimately extension of the DNA template would also fail to occur.